plants Brief Report First Report of Root Rot Caused by Phytophthora bilorbang on Olea europaea in Italy 1, 1,2,3, 2 2, Elena Santilli y, Mario Riolo y , Federico La Spada , Antonella Pane * and Santa Olga Cacciola 2,* 1 Council for Agricultural Research and Agricultural Economy Analysis, Research Centre for Olive, Citrus and Tree Fruit-Rende CS (CREA-OFA), 87036 Rende, Italy; [email protected] (E.S.); [email protected] (M.R.) 2 Department of Agriculture, Food and Environment, University of Catania, 95123 Catania, Italy; [email protected] 3 Department of Agricultural Science, Mediterranean University of Reggio Calabria, 89122 Reggio Calabria, Italy * Correspondence: [email protected] (A.P.); [email protected] (S.O.C.) These authors are equally contributed. y Received: 30 May 2020; Accepted: 27 June 2020; Published: 30 June 2020 Abstract: Leaf chlorosis, severe defoliation and wilt associated with root rot were observed on mature olive trees cv. Nera di Gonnos in an experimental orchard at Mirto Crosia (Calabria, southern Italy). An oomycete was consistently isolated from rotten roots of symptomatic olive trees. It was identified as Phytophthora bilorbang by morphological characters and sequencing of Internal Transcribed Spacer (ITS) regions of ribosomal DNA (rDNA). Pathogenicity was verified by inoculating potted two-month-old rooted cuttings of Olea europaea var. Nera di Gonnos in a soil infestation trial. P.bilorbang was re-isolated from roots of symptomatic, artificially inoculated olive cuttings to fulfill Koch’s postulates. This is the first report of P. bilorbang on O. europaea L. and on a species of the Oleaceae family worldwide. Keywords: root rot; Phytophthora; olive; leaf chlorosis; defoliation; wilt; molecular identification; morphological identification; ITS region 1. Introduction Olive (Olea europaea L., family Oleaceae) originated in the Near East and spread westward in the Mediterranean basin where it is widely cultivated as a fruit tree [1,2]. Based on data from the International Olive Oil Council (IOOC), more than 10 million hectares are cultivated with olive globally and 95% of them are in the Mediterranean basin [3]. With ca. 1.1 million ha of olive groves, Italy is the second largest olive growing country in the world, with Apulia, Calabria, and Sicily regions of southern Italy accounting for about 70% of the production [4]. As a typical Mediterranean plant, olive has been traditionally cultivated in arid lands. However, during the last decades in many olive growing countries, including Italy, the olive cultivation has been extended to different types of soil and irrigation has become a common practice in olive orchards. An emerging phytopathological problem of olive trees growing in wet or waterlogged soils is root rot caused by Phytophthora spp. [5]. The genus Phytophthora (Pythiaceae, Peronosporales, Oomycota, and Chromista) comprises more than 180 described taxa [6]. With the advent of DNA sequencing the systematics of the genus evolved from morphological criteria to molecular phylogeny. Therefore, the species of Phytophthora were identified using molecular markers and grouped into 12 phylogenetic clades some of which have subclades [7–10]. Several Phytophthora species of different phylogenetic clades have been reported as causative agents of leaf chlorosis, wilting, defoliation, and twig dieback, as a consequence of root rot and basal stem cankers Plants 2020, 9, 826; doi:10.3390/plants9070826 www.mdpi.com/journal/plants Plants 2020, 9, 826 2 of 9 on olive worldwide, including Phytophthora acerina, Phytophthora cactorum, Phytophthora cinnamomi, PhytophthoraPlants 20 citricola20, 9, x sensu lato, Phytophthora cryptogea, Phytophthora drechsleri, Phytophthora2 of inundata,9 Phytophthora megasperma, Phytophthora nicotianae, Phytophthora oleae, Phytophthora palmivora, Phytophthoraand basal pini, stemand cankersPhytophthora on olive worldwide, plurivora including[5,11–21 Phytophthora]. These speciesacerina, Phytophthora differ in aggressiveness,cactorum, Phytophthora cinnamomi, Phytophthora citricola sensu lato, Phytophthora cryptogea, Phytophthora drechsleri, temperature requirements for growth, geographical distribution, and ecology. Phytophthora palmivora, Phytophthora inundata, Phytophthora megasperma, Phytophthora nicotianae, Phytophthora oleae, alone orPhytophthora in association palmivora, with VerticilliumPhytophthora dahliaepini, and, wasPhytophthora reported plurivora as causal [5,11 agent–21]. These of rot species of fine differ roots in and wilt of youngaggressiveness, olive trees in temperature nurseries andrequirements new plantings for growth, in Italy geographical [12,22]. Phytophthora distribution, oleaeand ecology.was recovered from soilPhytophthora and roots palmivora of wild olive, alone trees or in inassociation protected with natural Verticillium areas dahliae in Spain, was and reported Sicily as (southern causal agent Italy) and is widespreadof rot of in fine soil roots of commercial and wilt of young olive orchardsolive trees in in Calabria nurseries (southernand new plantings Italy) [20 in,23 Italy,24 ].[12,2Phytophthora2]. inundataPhytophthorais responsible oleae was for recovered root rot from and soil wilt and of roots olive of treeswild olive in clay trees soils in protected after flooding, natural areas acting in as an Spain and Sicily (southern Italy) and is widespread in soil of commercial olive orchards in Calabria opportunistic(southern albeit Italy) aggressive [20,23,24]. Phytophthora root pathogen inundata [15, 18is responsibl,25]. In moiste for root environments, rot and wilt of some olive soil-inhabitingtrees in species,clay like soilsP. nicotianaeafter flooding,and actingP. oleae as anhave opportunistic occasionally albeit aggressive adapted toroot an pathogen aerial lifestyle[15,18,25]. and In moist may infect abovegroundenvironments, parts some of olive soil- treesinhabiting such species, as drupes, like P. nicotianae leaves, andand P. twigs oleae have causing occasionally fruit rot, adapted leaf drying, filloptosis,to an and aerial twig lifestyle dieback and may [26 –infect28]. aboveground parts of olive trees such as drupes, leaves, and twigs In autumncausing fruit 2019, rot, symptomsleaf drying, filloptosis of defoliation,, and twig wilt, dieback and root[26–2 rot8]. were observed on 15-year-old olive In autumn 2019, symptoms of defoliation, wilt, and root rot were observed on 15-year-old olive trees in an experimental orchard at Mirto Crosia, in Calabria (Figure1a). Trees were watered in summer trees in an experimental orchard at Mirto Crosia, in Calabria (Figure 1a). Trees were watered in using asummer drip irrigation using a drip system. irrigation About system. 40% About of the40% treesof the oftrees the of cv.the Neracv. Nera di di Gonnos, Gonnos, originating from Sardiniafrom (southern Sardinia Italy),(southern were Italy), aff ected.were affected. Symptoms Symptoms were were indicative indicative of Phytophthoraof Phytophthora root rot rot (PRR). The main(PRR) aim. The of themain present aim of the study present was study to identify was to identify the causative the causative agent agent of this of this disease. disease. Figure 1.Figure(a) 1. Decline (a) Decline symptoms symptoms on on a a tree tree ofof oliveolive ( (OleaOlea europaea europaea) cv.) cv.Nera Nera di Gonnos di Gonnos incited incitedby by PhytophthoraPhytophthora bilorbang bilorbangin Calabria. in Calabria. ( b(b)) WiltWilt of of a apotted potted rooted rooted cutting cutting of olive of cv. olive Nera cv.di Gonnos Nera di(on Gonnos (on thethe left) left) artificially artificially inoculated inoculated with P.P. bilorbang bilorbang throughthrough the thesoil, soil, 6 weeks 6 weeks after aftertransplanting transplanting into into infestedinfested soil, and soil, control and control non-inoculated non-inoculated cutting cutting (on (on the the right).right). ( (cc) )Morphology Morphology of 6 of-day 6-day-old-old colonies colonies of of P. bilorbang grown on V8 juice-agar and (d) on potato-dextrose-agar at 25 °C in the dark. P. bilorbang grown on V8 juice-agar and (d) on potato-dextrose-agar at 25 ◦C in the dark. 2. Results 2. Results 2.1. Species Identification and Morphological Features of Isolates 2.1. Species Identification and Morphological Features of Isolates Isolations from rotten roots of symptomatic olive trees sampled in the experimental orchard of IsolationsMirto Crosia from found rotten consistently roots of a symptomatic homothallic Phytophthora olive trees taxon sampled with a notable in the experimentalcolony morphology orchard of Mirto Crosia(Figure found 1c,d). consistentlyEighteen single a homothallic-hypha isolatesPhytophthora of this Phytophthtaxonora with taxon a notable, obtained colony from morphologythree (Figure1independentc,d). Eighteen trees single-hypha (six from each isolates tree), were of thischaracterized.Phytophthora Theytaxon, formed obtained stellate to from petaloid three colonies independent trees (sixon V8 from juice each-agar (V8A) tree), and were dense characterized.-felty, chrysanthemum They- formedlike and dome stellate shaped to in petaloid the center colonies colonies on V8 on potato-dextrose-agar (PDA). Extreme temperatures for growth were 4 (minimum) and 32 °C juice-agar (V8A) and dense-felty, chrysanthemum-like and dome shaped in the center colonies on (maximum), with an optimum at 25 °C. Sporangia formed on V8A were persistent, non-papillate, potato-dextrose-agar (PDA). Extreme temperatures for growth were 4 (minimum) and 32 ◦C (maximum),